An Herreman

Total inactivation of gamma-secretase activity in presenilin-deficient embryonic stem cells.

he question of how proteins such as the β-amyloid precursor protein (β-APP), Notch-1 and others can be cleaved in the plane of the membrane has challenged cell biologists and researchers into Alzheimer’s disease for some time. Processing of β-APP by γ-secretase is considered an important therapeutic target, as it constitutes the final step in the release of the amyloid βpeptide (Aβ), the principal constituent of the amyloid plaques in the brains of Alhzeimer’s patients. Recently, γ-secretase activity was linked to two proteins, presenilin-1 (PS1) and presenilin-2 (PS2; refs 1, 2). Presenilins are strongly hydrophobic proteins embedded in intracellular membranes, and missense mutations in their genes cause a rare, aggressive form of Alzheimer’s disease. It has been proposed that two aspartate residues in the transmembrane domains of presenilins constitute the catalytic site of γsecretase. One argument against this hypothesis is the important residual γ-secretase activity observed in cells derived from PS1or PS2-deficient mice. The question is therefore whether cells that are completely devoid of PS1 and PS2 maintain this γ-secretase activity or not. Unfortunately, PS-null mice die early in embryogenesis (after roughly 9.5 days of development; E9.5), making it impossible to generate sufficient cells to carry out the required biochemical experiments. We therefore generated pluripotent lines of embryonic stem cells from PS-null blastocysts obtained by mating of PS1PS2 mice. We genotyped the cell lines using the polymerase chain reaction (PCR) and Southern blotting (Fig. 1a). The 32% yield of double-deficient (PS-null) cell lines was slightly higher than the expected 25%; we confirmed the absence of PS1 (Fig. 1b) and PS2 (data not shown) by western blotting. We observed the accumulation of endogenous βand α-secretase-cleaved β-APP carboxy-terminal stubs, the direct substrates for γ-secretase (Fig. 1b and ref. 1). We then expressed β-APP695 harbouring the K595N/M596L (Swedish) mutation in the embryonic stem cells, using recombinant Semliki Forest virus (SFV). This mutation markedly increases production of Aβ , facilitating its detection in our assays. In contrast to results obtained with single-knockout cells, no Aβ was detected in the conditioned media of PS1PS2 cells (Fig. 1c). Using serial dilutions, we established that we would have detected Aβ even if its production was <0.5% of that in wildtype cells (Fig. 1c). We further confirmed the absence of γ-secretase activity using an enzyme-linked immunosorbent assay (ELISA) specific for the 42-amino-acid form of Aβ. mNotch∆E is another substrate for PS1-dependent γsecretase. Processing of this construct mimics that of full-length Notch-1, and results in release of the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates, together with transcription factors of the CBF-1 family, several genes, including HES-1 (ref. 9). We therefore co-transfected the embryonic stem cells with plasmids encoding mNotch∆E and luciferase under the control of the HES-1 promoter (kindly provided by R. Kopan and A. Israel), and measured induction of luciferase activity. As a control, we also transfected embryonic stem cells with plasmids encoding the NICD. Induction of HES-1 activity with mNotch∆E was observed in PS1PS2 cells, but not in PS1 PS2 cells (Fig. 1d). In contrast, NICD strongly induced luciferase activity, implying that the Notch signalling pathway downstream of the proteolytic processing of mNotch∆E was essentially intact in PS1PS2 cells. This was further confirmed by the observation that signalling in PS1PS2 cells was also restored after co-transfection with PS1 complementary DNA (data not shown). These observations extend to the molecular level our previous conclusion that the phenotype of PS1PS2 embryos is similar to that of Notch-deficient embryos. We further confirmed that proteolysis of mNotch∆E in PS1PS2 cells was completely inhibited using a biochemical assay, as before (Fig. 1e). Quantification of the obtained signals by phosphorimaging indicated that NICD production in PS1PS2 cells was <1% of that observed in PS1PS2 cells. In PS1PS2 cells, a weak NICD signal was already observed after 30 min of pulse labelling (Fig. 1f), whereas it was never observed in PS1PS2 cells. Studies involving overexpression of dominant-negative PS2 mutants have implicated PS2 in γ-secretase activity. However, here we have shown for the first time that γ-secretase activity is completely eliminated in embryonic stem cells that are devoid of presenilins. Although these results do not categorically prove that presenilins have catalytic activities, they constitute an important piece of indirect evidence in support of the ‘presenilin is γ-secretase’ hypothesis, by resolving one important counterargument. Our results also add to the emerging concept of ‘regulated intramembrane proteolysis’ (RIP). RIP cleaves integral membrane proteins in the plane of the membrane, releasing cytosolic fragments that enter the nucleus to control the transcription of genes involved in differentiation, lipid metabolism and the unfolded-protein response during endoplasmic reticulum stress. We anticipate that the cell lines generated in this study will greatly facilitate further investigation of the function of presenilins in RIP, and the identification of unknown presenilin substrates. h

Role of Presenilins in Neuronal Calcium Homeostasis

Alzheimers disease (AD) is a progressive and irreversible neurodegenerative disorder. Familial AD (FAD) mutations in presenilins have been linked to calcium (Ca2+) signaling abnormalities. To explain these results, we previously proposed that presenilins function as endoplasmic reticulum (ER) passive Ca2+ leak channels. To directly investigate the role of presenilins in neuronal ER Ca2+ homeostasis, we here performed a series of Ca2+ imaging experiments with primary neuronal cultures from conditional presenilin double-knock-out mice (PS1dTAG/dTAG , PS2 −/−) and from triple-transgenic AD mice (KI–PS1M146V , Thy1–APPKM670/671NL , Thy1–tauP301L ). Obtained results provided additional support to the hypothesis that presenilins function as ER Ca2+ leak channels in neurons. Interestingly, we discovered that presenilins play a major role in ER Ca2+ leak function in hippocampal but not in striatal neurons. We further discovered that, in hippocampal neurons, loss of presenilin-mediated ER Ca2+ leak function was compensated by an increase in expression and function of ryanodine receptors (RyanRs). Long-term feeding of the RyanR inhibitor dantrolene to amyloid precursor protein–presenilin-1 mice (Thy1–APPKM670/671NL , Thy1–PS1L166P ) resulted in an increased amyloid load, loss of synaptic markers, and neuronal atrophy in hippocampal and cortical regions. These results indicate that disruption of ER Ca2+ leak function of presenilins may play an important role in AD pathogenesis.

Deficiency of Aph1B/C-γ-secretase disturbs Nrg1 cleavage and sensorimotor gating that can be reversed with antipsychotic treatment

Regulated intramembrane proteolysis by γ-secretase cleaves proteins in their transmembrane domain and is involved in important signaling pathways. At least four different γ-secretase complexes have been identified, but little is known about their biological role and specificity. Previous work has demonstrated the involvement of the Aph1A-γ-secretase complex in Notch signaling, but no specific function could be assigned to Aph1B/C-γ-secretase. We demonstrate here that the Aph1B/C-γ-secretase complex is expressed in brain areas relevant to schizophrenia pathogenesis and that Aph1B/C deficiency causes pharmacological and behavioral abnormalities that can be reversed by antipsychotic drugs. At the molecular level we find accumulation of Nrg1 fragments in the brain of Aph1BC−/− mice. Our observations gain clinical relevance by the demonstration that a Val-to-Leu mutation in the Nrg1 transmembrane domain, associated with increased risk for schizophrenia, affects γ-secretase cleavage of Nrg1. This finding suggests that dysregulation of intramembrane proteolysis of Nrg1 could increase risk for schizophrenia and related disorders.

Notch Is a Critical Component of the Mouse Somitogenesis Oscillator and Is Essential for the Formation of the Somites

Segmentation of the vertebrate body axis is initiated through somitogenesis, whereby epithelial somites bud off in pairs periodically from the rostral end of the unsegmented presomitic mesoderm (PSM). The periodicity of somitogenesis is governed by a molecular oscillator that drives periodic waves of clock gene expression caudo-rostrally through the PSM with a periodicity that matches somite formation. To date the clock genes comprise components of the Notch, Wnt, and FGF pathways. The literature contains controversial reports as to the absolute role(s) of Notch signalling during the process of somite formation. Recent data in the zebrafish have suggested that the only role of Notch signalling is to synchronise clock gene oscillations across the PSM and that somite formation can continue in the absence of Notch activity. However, it is not clear in the mouse if an FGF/Wnt-based oscillator is sufficient to generate segmented structures, such as the somites, in the absence of all Notch activity. We have investigated the requirement for Notch signalling in the mouse somitogenesis clock by analysing embryos carrying a mutation in different components of the Notch pathway, such as Lunatic fringe (Lfng), Hes7, Rbpj, and presenilin1/presenilin2 (Psen1/Psen2), and by pharmacological blocking of the Notch pathway. In contrast to the fish studies, we show that mouse embryos lacking all Notch activity do not show oscillatory activity, as evidenced by the absence of waves of clock gene expression across the PSM, and they do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for the formation of a segmented body axis.

Gene expression profiling reveals clear differences between EBV-positive and EBV-negative posttransplant lymphoproliferative disorders

Posttransplant patients are at risk of developing a potentially life‐threatening posttransplantation lymphoproliferative disorder (PTLD), most often of diffuse large B cell lymphoma (DLBCL) morphology and associated with Epstein–Barr Virus (EBV) infection. The aim of this study was to characterize the clinicopathological and molecular‐genetic characteristics of posttransplant DLBCL and to elucidate whether EBV(+) and EBV(−) posttransplant DLBCL are biologically different. We performed gene expression profiling studies on 48 DLBCL of which 33 arose posttransplantation (PT‐DLBCL; 72% EBV+) and 15 in immunocompetent hosts (IC‐DLBCL; none EBV+). Unsupervised hierarchical analysis showed clustering of samples related to EBV‐status rather than immune status. Except for decreased T cell signaling these cases were inseparable from EBV(−) IC‐DLBCL. In contrast, a viral response signature clearly segregated EBV(+) PT‐DLBCL from EBV(−) PT‐DLBCL and IC‐DLBCL cases that were intermixed. The broad EBV latency profile (LMP1+/EBNA2+) was expressed in 59% of EBV(+) PT‐DLBCL and associated with a more elaborate inflammatory response compared to intermediate latency (LMP1+/EBNA2−). Inference analysis revealed a role for innate and tolerogenic immune responses (including VSIG4 and IDO1) in EBV(+) PT‐DLBCL. In conclusion we can state that the EBV signature is the most determining factor in the pathogenesis of EBV(+) PT‐DLBCL.

Clinicopathological characteristics of posttransplant lymphoproliferative disorders of T-cell origin: single-center series of nine cases and meta-analysis of 147 reported cases

Abstract T-cell or natural killer (NK)-cell posttransplant lymphoproliferative disorder (T-PTLD) is a rare but severe complication after transplant. Here we present the clinicopathological features of a single-center series of nine cases. Additionally, we summarize the clinicopathological findings of 147 cases of T/NK-cell PTLD reported in the literature in an attempt to define subtype-specific characteristics. T/NK-cell PTLD occurs in patients of all ages, usually extranodally, and most frequently after kidney transplant. Organ specific incidence, however, is highest following heart transplant. Approximately one-third of T-cell PTLDs are Epstein–Barr virus (EBV)-related, with peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) being the most prevalent EBV-associated T-cell PTLD. A male predominance is observed, which is most striking in the EBV(+) group, particularly in PTCL, NOS. With a median posttransplant interval of 72 months, T-cell PTLDs are among the late-occurring PTLDs. Of the most common T-cell PTLDs, anaplastic large cell lymphoma (ALCL) has the best prognosis, whereas PTCL, NOS and hepatosplenic T-cell lymphoma (HSTCL) have the worst prognosis. EBV(+) cases seem to have a longer survival than EBV(−) cases, suggesting a different pathogenetic mechanism.

The accuracy of positron emission tomography in the detection of posttransplant lymphoproliferative disorder

We investigated sensitivity, specificity, positive predictive value, negative predictive value and accuracy of 18F-fluorodeoxyglucose-positron emission tomography in 170 cases with suspected or biopsy-proven posttransplant lymphoproliferative disorder. All solid organ and hematopoietic stem cell transplant recipients who underwent an 18F-fluorodeoxyglucose-positron emission tomography scan between 2003 and 2010 in our center for the indication posttransplant lymphoproliferative disorder, were retrospectively reviewed and results were compared with tissue biopsy whenever possible. One hundred and seventy positron emission tomography scans in 150 patients were eligible for evaluation. In 45 cases, the patient had a biopsy-confirmed posttransplant lymphoproliferative disorder before positron emission tomography scanning and positron emission tomography was performed for staging purposes. In the remaining 125 cases, positron emission tomography was performed to differentiate between posttransplant lymphoproliferative disorder and other diseases. 18F-fluorodeoxyglucose-uptake was quantitatively expressed by calculation of maximum and mean standardized uptake value in the most intense lesion or, in the absence of attenuation corrected positron emission tomography scans, by comparing uptake in target lesion to liver and mediastinal uptake. We found an overall sensitivity of 89%, specificity of 89%, positive predictive value of 91% and negative predictive value of 87% for posttransplant lymphoproliferative disorder detection by 18F-fluorodeoxyglucose-positron emission tomography. In a subanalysis of the 125 scans performed for differentiating posttransplant lymphoproliferative disorder from other diseases, sensitivity, specificity, positive predictive value and negative predictive value were 90%, 89%, 85% and 93%, respectively. 18F-fluorodeoxyglucose-uptake in posttransplant lymphoproliferative disorder was generally high with a median mean and maximum standardized uptake value of 9.0 (range 2.0–18.6) and 17.4 (range 2.6–26.4). Posttransplant lymphoproliferative disorder often had an atypical presentation on positron emission tomography with high incidence of extranodal involvement. In conclusion, from these data, we can conclude that 18F-fluorodeoxyglucose-positron emission tomography is highly sensitive for detecting posttransplant lymphoproliferative disorder and has an excellent ability to differentiate posttransplant lymphoproliferative disorder from non-malignant diseases.

No endogenous A|[beta]| production in presenilin-deficient fibroblasts

NATURE CELL BIOLOGY VOL 4 JULY 2002 http://cellbio.nature.com E164 To the editor — Presenilins (PSs) are involved in the cleavage of integral membrane proteins, such as amyloid-β precursor protein (APP), Notch, ErbB-4 and CD44. The question as to whether or not they are proteases is therefore relevant for the broad field of cell biological research. Recently, Armogida et al. reported that small amounts of the Aβ amyloid peptide can be detected in mouse PS-deficient fibroblasts. This result challenged the conclusions of previous publications. However, the assay used by Armogida et al. is not specific for mouse Aβ, and we were not able to reproduce their findings using a mouse Aβ-specific ELISA assay (Fig. 1a). When we used a similar immune precipitation/western blotting assay as Armogida et al., we did however detect trace amounts of Aβ peptide, even in the unconditioned culture medium (Fig. 1b). This assay is critically dependent on the monoclonal antibody, WO-2, which was raised against a specific human Aβ epitope. As both rabbit and bovine Aβ, but not mouse Aβ, are identical to human Aβ, they will be equally and efficiently detected by the WO-2 antibody. Thus, bovine Aβ from the serum added to the culture medium or rabbit Aβ (see accompanying letter by Grimm et al.) in the sera used to immunoprecipitate Aβ are potential sources of false-positive results. Although it is possible to incubate fibroblasts for brief periods in serum-free media (see accompanying letter by Petit et al.), it remains uncertain whether the bovine Aβ that accumulated before can be completely depleted from the cells. In any event, the affinity of the WO-2 antibody is too low to detect mouse Aβ from cells that overexpress mouse APP in this assay (Fig. 1c), demonstrating unequivocally that the Aβ detected in the experiments of Armogida et al. cannot be of mouse origin. Thus, the claim that PSdeficient fibroblasts secrete Aβ is not supported by the published data. A more extensive pdf report will be made available on request.

Macrophages in T cell/histiocyte rich large B cell lymphoma strongly express metal-binding proteins and show a bi-activated phenotype.

Abundant macrophage infiltration in tumors often correlates with a poor prognosis. T cell/histiocyte rich large B cell lymphoma (THRLBCL) is a distinct aggressive B cell lymphoma entity showing a high macrophage content. To further elucidate the role of tumor‐associated macrophages in THRLBCL, we performed gene expression profiling of microdissected histiocyte subsets of THRLBCL, nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), Piringer lymphadenitis, sarcoidosis, nonspecific lymphadenitis and monocytes from peripheral blood. In a supervised principal component analysis, histiocytes from THRLBCL were most closely related to epithelioid cells from NLPHL, with both types of cells expressing genes related to proinflammatory and regulatory macrophage activity. Moreover, histiocytes from THRLBCL strongly expressed metal‐binding proteins like MT2A, by which histiocytes of THRLBCL can be distinguished from the other histiocyte subsets investigated. Interestingly, the validation at the protein level showed a strong expression of TXN, CXCL9, MT2A and SOD2 not only in macrophages of THRLBCL but also in the tumor cells of NLPHL and classical Hodgkin lymphoma (cHL). Overall, the present findings indicate that macrophages in the microenvironment of THRLBCL have acquired a distinct gene expression pattern that is characterized by a mixed M1/M2 phenotype and a strong expression of several metal binding proteins. The microenvironments in NLPHL and THRLBCL appear to have a similar influence on the macrophage phenotype. The high expression of metal binding proteins in histiocytes of THRLBCL may be diagnostically useful, but a potential pathophysiological role remains to be identified.

21-P047 Notch is essential for the mouse segmentation clock

We first cloned Xenopus Rheb, which is generally believed as the upstream positive regulator of TOR, then created dominant negative form of Rheb (dn-Rheb), and also designed antisense morpholino oligomers of Rheb (Rheb-MO). Gain-of-functional experiment of TOR signal by microinjection of Rheb mRNA into blastomeres led the posterior defects for the embryos. FKBP38 is the direct negative regulator of TOR, so FKBP38-MO was also designed. Similar to the case of Rheb mRNA, FKBP38-MO injected embryos lost posterior structures. On the other hand, loss-offunctional experiment by microinjection of dn-Rheb or RhebMO into dorsal blastomeres strongly reduced the size of head structure, and no additional posterior structures were observed. Taken together with these results, we conclude that anteroposterior positional value of Xenopus early development is determined by the level of TOR signal. We also did loss-of-functional experiment of TOR signal using rapamycin that directly binds to TOR and inhibit its activity. Interestingly, rapamycin treated embryos were losing pigmentation derived from neural crest and pronephros with unusual stomach and gut formation, which is also reported in zebrafish studies.